Heterocyclic compounds, combinatorial libraries thereof and methods of selecting drug leads

ABSTRACT

Heterocyclic compounds having a relatively flexible backbone are used to create combinatorial libraries that permit screening for lead compounds and selection of drug candidates for a variety of uses in human and veterinary medicine as well as in agriculture. The compounds of the library generally differ in ring size and chirality of substituents on the ring. Also disclosed are methods for providing and screening these libraries, preferably in an automated or computerizable manner, such as by using a computer program to virtually screen the compounds in order to identify those that are predicted to have bioactive conformations that should give rise to desirable biological effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/882,636filed Jul. 2, 2004, which is a continuation of International applicationPCT/IL03/00008 filed Jan. 2, 2003, which in turn is a continuation ofapplication Ser. No. 10/034,312 filed Jan. 3, 2002, the entire contentof each of which is expressly incorporated herein by reference thereto.

FIELD OF THE INVENTION

This invention is generally in the field of combinatorial chemistry anduse of combinatorial libraries of heterocyclic compounds to select anddevelop new drugs. More specifically, the present invention providesnovel heterocyclic compounds that have are relatively flexible backbone,combinatorial libraries comprising these compounds, which may be used toscreen for and to select drug candidates for a variety of uses in humanmedicine, veterinary medicine and in agriculture.

BACKGROUND OF THE INVENTION

An important objective of combinatorial chemistry is to generate a largenumber of novel compounds that can be screened to identify leadcompounds for pharmaceutical research and drug development.Theoretically, the total number of compounds which may be produced for agiven library is limited only by the number of reagents available toform substituents on the variable positions on the library's molecularscaffold. The combinatorial process lends itself to automation, both inthe generation of compounds and in their biological screening, therebyenhancing greatly the opportunity and efficiency of drug discovery.

Combinatorial chemistry may be performed in a manner where libraries ofcompounds are generated as mixtures, while the complete identificationof the individual compounds is postponed until after positive screeningresults are obtained. However, a preferred form of combinatorialchemistry is “parallel array synthesis”, (also called Multiple ParallelSynthesis, MPS) where individual reaction products are simultaneouslysynthesized, but are retained in separate compartments [Geysen et al.(1984); Houghten (1985)]. For example, the individual library compoundscan be prepared, stored, and assayed in separate wells of a microtiterplate, each well containing one member of the parallel array. The use ofstandardized microtiter plates or equivalent apparatus is advantageousbecause such an apparatus is readily accessed by programmed roboticmachinery, both during library synthesis and during library sampling orassaying.

Combinatorial chemistry can be carried out in solution phase where bothreactants are dissolved in solution or in solid phase where one of thereactants is covalently bound to a solid support. Solid phase synthesisoffers the advantage that reactions can be carried out using excessreagents, while the solid support-bound products are easily washed freeof excess reagent. The use of excess reagents may ensure high yield ofeach step in a multiple step synthesis. Solution phase synthesistypically requires use of one or more reaction mixture work-upprocedures to separate reaction product from unreacted excess reagent.

The first combinatorial libraries were composed of peptides, in whichall or selected amino acid positions were randomized [Geysen et al.(1984); Furka et al. (1991)]. Peptides and proteins can exhibit high andspecific binding activity, and can act as catalysts. In consequence,they are of great importance in biological systems. Unfortunately,peptides per se have limited utility for use as therapeutic entities.They are costly to synthesize, unstable in the presence of proteases,non selective and in general do not pass cellular membranes.

Nucleic acids have also been used in combinatorial libraries. Theirgreat advantage is the ease with which a nucleic acid with appropriatebinding activity can be amplified. As a result, combinatorial librariescomposed of nucleic acids can be of low redundancy and hence, of highdiversity. However, the resulting oligonucleotides are not suitable asdrugs for several reasons. First, the oligonucleotides have highmolecular weights and cannot be synthesized conveniently in largequantities. Second, because oligonucleotides are polyanions, they do notcross cell membranes. Finally, deoxy- and ribo-nucleotides arehydrolytically digested by nucleases that occur in all living systemsand are therefore usually decomposed before reaching the target.

There has therefore been much interest in combinatorial libraries basedon small molecules (i.e. molecules having molecular weight of up toabout 1000 daltons), which are more suited to pharmaceutical use,especially those which, like benzodiazepines, belong to a chemical classwhich has already yielded useful pharmacological agents [Bunin andEllman (1992); Beeley (2000)]. The techniques of combinatorial chemistryhave been recognized as the most efficient means for finding smallmolecules that act on these targets. At present, small moleculecombinatorial chemistry involves the synthesis of either pooled ordiscrete molecules that present varying arrays of functionality on acommon scaffold. These compounds are grouped in libraries that are thenscreened against the target of interest either for binding or forinhibition of biological activity [Adang and Hermkens (2001)].

The elements of diversity in libraries of currently available scaffoldbased compounds having the general structure (A) shown below, are basedmainly on sequential or positional diversity namely the order in whichthe various R groups are arranged around the ring and chemicaldiversity-that can arise from alterations in the chemical nature of theR groups.

In the above structure (A), X, Y and Z represent ring heteroatoms orcarbons, and R′, R″ and R′″ represent substituents associated to thering through a linker (showed schematically as a wavy line).

It is known from the art [Kumar S. et al. (2000)] that molecules maybind to each other if their conformations are complementary in geometryand chemistry and if their binding produces stable associations.However, most of the known libraries of organic molecules suffer from amajor drawback when applied for the discovery of new drug leads based onthe inhibition of peptide: protein or protein: protein or protein:nucleic acid interactions: they are too constrained and therefore lackthe ability to undergo conformational complementarity, i.e. lack anability for binding to a protein and/or a nucleic acid. This led to thepreparations of extremely large libraries (consist of up to millions ofcompounds) and their biological screening, which in many cases resultsin the discovery of low affinity leads or to the lack of theirdiscovery.

There is thus an urgent need in the art to develop new combinatoriallibraries comprising molecules having a flexible scaffold backbone thatare conformationally flexible and thus have the ability to undergoconformational complementarity. Such libraries will be useful in thescreening for drug candidates for a variety of uses in medicine.

SUMMARY OF THE INVENTION

The present invention provides, according to a first aspect, novelheterocyclic compounds that have a relatively flexible backbone.According to another aspect of the present invention, these compoundsmay be used to produce new combinatorial libraries that will serve interalia in high throughput screening assays, to screen for and select drugcandidates for a variety of uses in human medicine, veterinary medicineand in is agriculture.

The present invention further provides combinatorial libraries ofheterocyclic compounds having a ring size of between 8 to 20 atomswherein the members of libraries provided by the invention differ fromeach other (in addition to the conventional chemical and positionaldiversity attained by the different substituents on the ring) in atleast one of two novel aspects: (a) the ring size; and/ or (b) thechirality of the substituents on the ring. This leads to conformationaldiversity and flexibility that allows the selection of the most activecompound, not only on the basis of the nature and arrangement of thesubstituents (attained by the known chemical and positional diversity),but also based on the ability to undergo conformational complementarity(attained by the conformational diversity and/or the chiral diversity).The present invention further provides combinatorial librariescomprising a plurality of the heterocyclic compounds of the presentinvention, and methods, including computerized methods, of screening thecombinatorial libraries for compounds having a desired beneficialbiological effect.

The present invention further provides, in accordance with anotheraspect, a combinatorial library comprising a plurality of heterocycliccompounds, wherein the ring size of the heterocyclic compounds isbetween 8 and 20 atoms, and members of the library contain at least onecommon pharmacophore but differ from each other by at least one of:

-   -   (i) the size of the ring; and (ii) the chirality of the        substituents on the ring.

In addition to the parameters above, preferably the members of thelibrary may differ from each other by at least one additional parameterselected from:

-   -   (iii) the chemical nature of the ring;    -   (iv) the order of the substituents on the ring;    -   (v) the type of linker connecting the substituent to the ring.

Preferably, each member of the library bears at least two, morepreferably at least three, yet more preferably at least foursubstituents. Typically the at least two substituents are independentlyselected from: hydrogen, a linear or branched chain alkyl, cycloalkyl,aryl, aralkyl, heterocyclyl, heteroaryl, acyl, carboxyalkyl,carboxyaryl, benzyl, hydroxybenzyl, benzyloxycarbonyl, a side chain of anatural or an unnatural amino acid or a peptide.

Within the scope of this specification and the claims which follow theterm “pharmacophore” denotes any three dimensional array of descriptorswhich may be used to represent the therapeutic or other biologicalactivity of a given molecule. Descriptors are expressed in terms ofinteractions observed in molecular recognition including but not limitedto hydrogen bonding, ionic interactions, electrostatic field, van derWaals interactions, and hydrophobic interactions. The pharmacophore, maythus be defined as a model of the three dimensional orientation of a setof features which describe the physical, chemical and electronicenvironment of a set of molecules exerting the specific biologicalactivity, said features comprising for example the hydrogen bond donorfeature, the hydrogen bond acceptor feature, the hydrophobic regionfeature, the ionizable region feature, the aromatic ring feature. Thethree dimensional structure of the target molecule, which iscomplementary to the pharmacophore, can be represented by a coordinatesystem defining the positions of the amino acids or other molecularfeatures interacting with said pharmacophore. The pharmacophore or itscomplementary target may be represented by a coordinate system that isconfigured in a computer readable format.

Thus, in one embodiment, the present invention provides a heterocycliccompound represented by the structure of formula I:

wherein

-   -   A, B and D are independently of each other CH₂, C═O or a bond;    -   X and Y are independently of each other O, S, C═O, S═O, SO₂,        CR_(3a)R_(3b), NR₄ or C═S, or X and Y together form a group        represented by the formula:        R_(1a), R_(1b), R_(2a), R_(2b), R_(3a), R_(3b), R_(4a) and        R_(4b) are independently of each other hydrogen or a linear or        branched chain alkyl;    -   m and n are independently of each other an integer of 1-6;    -   Z and Q are independently of each other        R_(5a), R_(5b), R_(6a), R_(6b), R_(7a), R_(7b), R₈, R_(9a), and        R_(9b) are independently of each other hydrogen, a linear or        branched chain alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,        heteroaryl, acyl, carboxyalkyl, carboxyaryl, benzyl,        hydroxybenzyl, benzyloxycarbonyl, a side chain of a natural or        unnatural amino acid or a peptide;    -   L is hydrogen, OR₁₀, or NHR₁₁ wherein R₁₀ and R₁₁ are        independently of each other hydrogen, a linear or branched chain        alkyl, a side chain of a natural or unnatural amino acid, a        peptide or a solid support; and        is a heterocyclic moiety containing one or more nitrogens;    -   or a pharmaceutically acceptable salt, hydrate or solvate        thereof.

In one embodiment, the present invention provides a compound of formulaI wherein A and D are a bond and B is C═O. In another embodiment, thepresent invention provides a compound of formula I wherein A is a bond,B is CH₂ are C is C═O.

In another embodiment, the present invention provides a compound offormula I wherein Q is

In another embodiment, the present invention provides a compound offormula I wherein Z is

In another embodiment, the present invention provides a compound offormula I wherein one of R_(5a) and R_(5b) is hydrogen and the other isbenzyl. In another embodiment, the present invention provides a compoundof formula I wherein one of R_(5a) and R_(5b) is hydrogen and the otheris hydroxybenzyl. In another embodiment, the present invention providesa compound of formula I wherein one of R_(6a) and R_(6b) is hydrogen andthe other is benzyl. In another embodiment, the present inventionprovides a compound of formula I wherein one of R_(6a) and R_(6b) ishydrogen and the other is hydroxybenzyl. In another embodiment, thepresent invention provides a compound of formula I wherein one of R_(7a)and R_(7b) is hydrogen and the other is benzyl. In another embodiment,the present invention provides a compound of formula I wherein one ofR_(7a) and R_(7b) is hydrogen and the other is hydroxybenzyl. In anotherembodiment, the present invention provides a compound of formula Iwherein R₈ is benzyloxycarbonyl. In another embodiment, the presentinvention provides a compound of formula I wherein one of R_(9a) andR_(9b) is hydrogen and the other is benzyloxycarbonyl. In anotherembodiment, the present invention provides a compound of formula Iwherein L is NH₂.

In another embodiment, the present invention provides a heterocycliccompound to represented by the structure of any of formulas II-X.

In one embodiment, the present invention provides a compound of formulaII. In another embodiment, the present invention provides a compound offormula III. In another embodiment, the present invention provides acompound of formula IV. In another embodiment, the present inventionprovides a compound of formula V. In another embodiment, the presentinvention provides a compound of formula VI. In another embodiment, thepresent invention provides a compound of formula VII. In anotherembodiment, the present invention provides a compound of formula VIII.In another embodiment, the present invention provides a compound offormula IX. In another embodiment, the present invention provides acompound of formula X.

In one embodiment, the compound of any of formulas I-X comprises atleast one pharmacophore potentially associated with a biologicalactivity. In a preferred embodiment, the biological activity is mediatedby a cellular component. In another preferred embodiment, the cellularcomponent is a nucleic acid. In a particularly preferred embodiment, thecellular component is a protein. In yet another embodiment, thebiological activity is proliferation, differentiation, phenotypealteration, uptake of substances by cells, secretion of substances fromcells, metabolism, gene expression, protein expression, or anycombination thereof.

This invention also provides, according to another of its embodiments, acombinatorial library comprising a plurality of compounds represented bythe structure of any of formulas I-X. In one embodiment, the librarycomprises a plurality of compounds represented by the structure offormula I. In another embodiment, the library comprises a plurality ofcompounds represented by the structure of formula II. In anotherembodiment, the library comprises a plurality of compounds representedby the structure of formula III. In another embodiment, the librarycomprises a plurality of compounds represented by the structure offormula IV. In another embodiment, the library comprises a plurality ofcompounds represented by the structure of formula V. In anotherembodiment, the library comprises a plurality of compounds representedby the structure of formula VI. In another embodiment, the librarycomprises a plurality of compounds represented by the structure offormula VII. In another embodiment, the library comprises a plurality ofcompounds represented by the structure of formula VIII. In anotherembodiment, the library comprises a plurality of compounds representedby the structure of formula IX. In another embodiment, the librarycomprises a plurality of compounds represented by the structure offormula X. In another embodiment the library comprises a plurality ofcompounds represented by a plurality of structures selected fromformulas I through X.

In a preferred embodiment of the present invention, some members of thelibrary differ from the others by the size of the ring. In anotherpreferred embodiment, some members of the library differ from the otherby the chirality of the substituents on the ring. In another embodiment,some members of the library differ from the others by at least one ofthe size of the ring or the chirality of the substituents of the ring;and further differs by at least one of the chemical nature of the ring,the substituents on the ring, the linkers connecting the substituentsand the ring, the arrangement of the substituents on the ring, or anycombination thereof.

The combinatorial libraries of the invention serve as a readilyaccessible source of diverse macrocyclic compounds for use inidentifying new biologically active macrocyclic compounds throughpharmaceutical and veterinary candidate screening assays, for thedevelopment of lead candidates for pharmaceutical purposes, fordeveloping highly effective and environmentally friendly insect controland crop control agents, for use in studies defining structure/activityrelationships, and/or for use in clinical investigation.

Furthermore, in another embodiment, the present invention providesmethods for designing new compound libraries that have a novel type ofstructural complexity and diversity, and that can be screened toidentify potent compounds for pharmaceutical, veterinary or agriculturaluse. Thus, in one embodiment, the present invention provides a method ofidentifying a compound having a beneficial biological activity, by (a)designing a combinatorial library comprising a plurality of heterocycliccompounds having a ring size of between 8 to 20 atoms wherein somemembers of the library differ from others in at least one aspectselected from the ring size and the chirality of the substituents on thering, wherein each member of the library comprises at least onepharmacophore potentially associated with the biological activity; (b)synthesizing a plurality of compounds from the combinatorial library;and (c) screening the synthesized compounds for candidates having thedesired biological activity.

In one embodiment, the biological activity is achieved by modulation ofa cellular component. In one embodiment, the pharmacophore iscomplementary to a domain in the cellular component, which is associatedwith the biological activity so that the pharmacophore can bind to thedomain on the cellular component and thus change the biological activityassociated with the cellular component.

In accordance with a preferred embodiment the pharmacophore mimics adomain on a cellular component and thus interacts with another cellularcomponent in lieu of the interaction with the domain it mimics. This mayserve either as a “decoy” to eliminate the biological activityassociated with the cellular component or alternatively may serve as afunctional mimic of the cellular component and thus provide or enhancethe biological activity normally associated with the cellular component.

In one embodiment, the method further comprises the following steps aspart of the designing step (a): (a1) identifying a domain in a cellularcomponent which is associated with the biological activity, and (a2)virtually screening the combinatorial library for lead compounds havinga pharmacophore, which is capable of mimicking or is complementary tothe domain. In one embodiment, the virtual screening step comprisesvirtual screening of the library with a computer readable data storagematerial encoded with computer readable data comprisingthree-dimensional structural determinants defining the desired domain.In yet another aspect, the computer readable data storage material isfurther encoded with a computer program logic for controlling aprocessor, wherein the computer program logic comprising a procedurethat enables the processor to identify a member of the combinatoriallibrary having a specified pharmacophore.

Furthermore, in yet another embodiment, the present invention provides acomputer program product for virtual screening of the combinatoriallibrary of heterocyclic compounds provided herein for a compound havinga beneficial biological activity, the computer program productcomprising: (a) a computer readable data storage material encoded withcomputer readable data comprising three-dimensional structuraldeterminants defining a domain in a cellular component which isassociated with the desired biological activity; and (b) a computerprogram logic for controlling a processor, comprising a procedure thatenables the processor to identify a compound having a pharmacophore tothe desired domain.

Furthermore, in yet another embodiment, the present invention provides asystem for identifying a compound having a beneficial biological,comprising: (a) an automated device for virtual screening of thecombinatorial library of heterocyclic compounds provided herein for acompound having said beneficial biological activity; and (b) anexperimental device for screening these compounds for candidates havingthe desired biological activity.

In one embodiment, the automated device comprises the computer programproduct described hereinabove. In another embodiment, the experimentaldevice comprises (a) an apparatus for synthesizing the compoundsidentified in step (a); and (b) an apparatus for experimentallyscreening the compounds for candidates having the desired biologicalactivity.

Furthermore, in another embodiment, the present invention provides apharmaceutical composition comprising at least one heterocyclic compoundas defined hereinabove, and a pharmaceutically acceptable carrier.Furthermore, in yet another embodiment, the present invention provides amethod for the treatment of a disease, condition or disorder in asubject in need thereof, comprising the step of administering to thesubject a therapeutically effective amount of a heterocyclic compoundprovided by the present invention.

In one embodiment, the compounds defined by the present invention areuseful in the treatment of a disease, disorder or condition. In anotherembodiment, therapy of the disease, disorder or condition is achieved bymodulation of a cellular component, for example a protein, a peptide, anucleic acid-cellular component or a combination thereof. The compoundsprovided herein, either alone or in the pharmaceutical compositions aresuitable for use in any subject, for example a mammalian subject or ahuman subject. The pharmaceutical compositions of the present inventionare also suitable for use in veterinary medicine and furthermore may beused in agriculture.

The libraries of the new compounds of the present invention comprise anovel element of diversity, namely spatial diversity, that results fromthe varying size of the scaffold ring, the chirality of the linker orfrom a combination of the two. This diversity is new in the field ofsmall molecule combinatorial chemistry. Thus the compounds of thepresent invention have a flexible backbone that enables conformationalflexibility and accordingly these compounds have the ability to undergoconformational complementarity. The conformational complementarilyenables the compound of the invention to bind to cellular targets,modulate the biological activity associated by the target and thus causea physiological effect. Thus, the compounds of the present invention arepotentially useful as drug candidates for a variety of uses in medicine.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully fromthe following detailed description taken in conjunction with theappended drawings in which:

FIG. 1: shows structural super positioning of “SIB7”, a heterocycliccompound in accordance with the invention, and the activation loop ofIGF-1R;

FIG. 2: shows a FlexX space filed model of SIB7 docking into thesubstrate binding site of IGF-1R;

FIG. 3A: shows the phosphorylation levels of IGF-1R in the presence andabsence of SIB7;

FIG. 3B: shows the phosphorylation levels of ERK, a downstream elementof IGF1R, in the presence and absence of SIB7;

FIG. 4: shows dose dependent inhibition of MCF-7 breast cancer cellproliferation in the presence of SIB 7.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new heterocyclic compounds that have arelatively flexible backbone. These compounds may be used to produce newcombinatorial libraries that are useful, in methods including but notlimited to high throughput screening assays, to screen for and selectdrug candidates for a variety of uses in human medicine, veterinarymedicine and in agriculture.

The present invention further provides novel, ring based combinatoriallibraries, comprising heterocylic compounds having a ring size ofbetween 8-20 atoms wherein the members of libraries provided by theinvention differ from each other (in addition to the conventionalchemical and positional diversity attained by the different substituentson the ring) in two novel aspects: (a) the ring size; and (b) thechirality of the substitutents on the ring. This leads to conformationaldiversity and flexibility that allows the selection of the most activecompound, not only on the basis of the nature and arrangement of thesubstituents (attained by the known chemical and positional diversity),but also based on the ability to undergo conformational complementarity(attained by the conformational diversity and chiral diversity). Thepresent invention further provides combinatorial libraries comprising aplurality of the heterocyclic compounds of the present invention, andmethods for screening the combinatorial libraries for compounds having abeneficial biological effect. The screening methods provided herein maybe automated and/or computerized, for example by using a computerprogram to virtually screen the combinatorial libraries in order toidentify compounds that are predicted to adopt bioactive conformationsthat will give rise to the desired biological effect.

Many biological processes are critically dependent on protein:proteinand/or protein:nucleic acid interactions, and many drugs are smallmolecules known to disrupt such interactions (antagonists) oralternatively mimic one component of the interaction in such a manner sothat the activity controlled by the interaction can take place in thepresence of the drug and the other cellular component (agonists). Thedrugs, which work by interruption of such interactions (for example bythe interruption of a receptor-ligand interaction) mimic a domain of oneof the participants in the interaction (for example a protein).

In one case this mimic creates an antagonist that competes with theprotein for binding with the other member of the interaction (the othercellular component), leading to a decrease in the interaction and hencea decrease of the cellular activity controlled or caused (directly orindirectly) by the interaction. Where the cellular activity is an “on”physiological process, (for example, increase in production of anagent), the interruption will block the “on” reaction and decrease thephysiological process (resulting in a decrease in the production of theagent). Where the cellular activity is an “off” reaction (for example asignal causing inhibition of proliferation), the interruption will blockthe “off” reaction and will increase the physiological process, forexample, cause increased proliferation.

Alternatively, a drug may work as an agonist and cause the modulation ofthe cell activity by mimicking the protein (that is essential for thecell activity) in the interaction in such a manner that the cellactivity takes place as if the native protein and not the compound wereinteracting with the other cellular component. For example the compoundmay be able to activate the cellular component with which the proteininteracts in a similar way to the protein itself.

The aim of the compounds of the present invention is to mimic a regionin one of the participators of the interaction, so as to compete for thebinding on the other participator of the interaction (either in theantagonist or the agonist manner), thus changing the interaction andleading to a change in the physiological process or property controlledby the interaction.

The rationale for the present invention is the following: many librariesused for the discovery of drug leads are composed of heterocyclicscaffolds that are too constrained (rigid) to allow conformationalcomplementarity essential for the interactions with proteins, peptides,polysaccharides or nucleic acids. The combinatorial library of theinvention allows the generation of sub-libraries with spatial diversity,which is obtained by the diversity in ring size and chirality of thelink between the substituent and the ring backbone or by combinationsthereof. This results in a library where each individual member has adifferent flexibility and a different spatial positioning of thepharmacophore. The design of the library of the invention increases theprobability that some members of the library have the ability to undergoconformational complementarily, i.e. the pharmacophores are present inthe correct orientation to interrupt or mimic the interaction with theother cellular component. The present invention allows the design andsynthesis of libraries, which occupy a larger proportion of the“probability space” of the pharmacophore positioning (i.e. increase theprobability of the substituents to be present in varying positions inthe space, thus increasing the probability that at least onepositioning-conformation is the bioactive conformation), while stillcreating relatively small, focused libraries. These properties lead tofast discovery and optimization of novel drug leads.

The classical elements of diversity of state-of-the-art, currentlyavailable macrocyclic, i.e. scaffold based libraries are based mainlyon:

-   -   (1) The chemical nature of the scaffold (i.e. backbone);    -   (2) The size and chemical nature of the linkers that connect        between the backbone and the various substituents;    -   (3) The chemical nature of the substituents; and    -   (4) The order in which the substituents are arranged on the        ring.

The libraries of the new compounds of the present invention comprise anovel element of diversity, namely spatial diversity, that results from(a) the varying size of the ring, (b), the chirality of the linker; or(c) or a combination of the two. This diversity is new in the field ofsmall molecule combinatorial chemistry. In a preferred embodiment of thepresent invention, each member of the library differs from the other bythe size of the ring. In another preferred embodiment, each member ofthe library differs from the other by the chirality of the substituentson the ring. Also encompassed within the scope of the present inventionare libraries wherein each member differs from the other by both thesize of the ring and the chirality of the substituent of the ring.Furthermore, the members of the library may further differ from oneanother by the “conventional” elements of diversity (1-4 definedhereinabove), i.e. by at least one parameter selected from: the chemicalnature of the ring, the substituents on the ring, the linkers connectingthe substituents and the ring, the arrangement of the substituents onthe ring, or any combination thereof.

Spatial diversity is defined as diversity elements that alter theconformation of the compounds, which in fact lead to altered spatialpositioning of the pharmacophore or pharmacophores.

According to one specific embodiment of the present invention, themembers in a library share at least some of the elements defined initems (1)-(4) mentioned above: the same type of scaffold backbone withthe same composition of atoms within the ring; the same linker with thesame size and chemical nature; the same substituents arranged in thesame order on the ring, but they differ from each other in the size ofthe scaffold and/or the chirality of the linker. This in turn determinesthe possible conformational (spatial) positioning of the pharmacophoreof each compound and allows for the selection of the lead compoundhaving the appropriate ability of conformational complementarity. Thelibraries of the invention are composed of a series of compounds thatdiffer from each other by an incremental alteration of their possibleconformations. Thus, every library covers an entire range of theconformational probabilities and increases the chances of obtaining acompound with at least one bioactive conformation.

A “lead compound” is a library compound in a selected combinatoriallibrary, for which the assay has revealed significant effect relevant toa desired cell activity to be modulated. In the present case theproperty is the modulation of at least one biological activityassociated with a cellular component which the library compound eitherinteracts with or mimics.

The selection of an active candidate is preferably achieved from alibrary of compounds that have the same substituents in differentpositions along the ring but the rings differ from each other in sizeand chirality of the substituents and therefore in their conformation.The libraries are prepared by the multiple simultaneous solid phasemethod [Houghten R. A., 1985] or its automated version, and contain thecalculated number of diversity possibilities. Libraries are typicallysynthesized in a 12-48 format, namely each library typically contains12-48 members. Each member of the library will be characterized,purified and subjected to biological assay.

The pharmacophore has proven to be a highly valuable and useful conceptin drug discovery and drug-lead optimization. A pharmacophore is definedas a distinct three dimensional (3D) arrangement of chemical groupsessential for biological activity. Since a pharmaceutically activemolecule must interact with one or more molecular structures within thebody of the subject in order to be effective, and the desired functionalproperties of the molecule are derived from these interactions, eachactive compound must contain a distinct arrangement of chemical groupswhich enable this interaction to occur. The chemical groups, commonlytermed descriptor centers, can be represented by (a) an atom or group ofatoms; (b) pseudo-atoms, for example a center of a ring, or the centerof mass of a molecule; (c) vectors, for example atomic pairs, electronlone pair directions, or the normal to a plane. Clearly, the ability todesign, or identify from large databases, pharmaceutically usefulmolecules according to the pharmacophore would be highly effective bothin the process of drug discovery and in the process of drug leadoptimization.

In the present invention the term refers to those moieties of the sidechain or backbone of the cellular components, for example peptide,protein or nucleic acid (which mediate a cell activity), that arenecessary for the binding to the other cellular components, the bindingeliciting a biological response. The pharmacophore may be a chemicalmoiety present on a single side chain or a collection of chemicalmoieties present in spatially adjacent side chains.

Novel Compounds

Thus, in one embodiment, the present invention provides a heterocycliccompound represented by the structure of formula I.

wherein

-   -   A, B and D are independently of each other CH₂, C═O or a bond;    -   X and Y are independently of each other O, S, C═O, S═O, SO₂,        CR_(3a)R_(3b), NR₄ or C═S, or X and Y together form a group        represented by the formula:        R_(1a), R_(1b), R_(2a), R_(2b), R_(3a), R_(3b), R_(4a) and        R_(4b) are independently of each other hydrogen or a linear or        branched chain alkyl;    -   m and n are independently of each other an integer of 1-6;    -   Z and Q are independently of each other        R_(5a), R_(5b), R_(6a), R_(6b), R_(7a), R_(7b), R₈, R_(9a), and        R_(9b) are independently of each other hydrogen, a linear or        branched chain alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,        heteroaryl, acyl, carboxyalkyl, carboxyaryl, benzyl,        hydroxybenzyl, benzyloxycarbonyl, a side chain of a natural or        unnatural amino acid or a peptide;    -   L is hydrogen, OR₁₀, or NHR₁₁ wherein R₁₀ and R₁₁ are        independently of each other hydrogen, a linear or branched chain        alkyl, a side chain of a natural or unnatural amino acid, a        peptide or a solid support; and        is a heterocyclic moiety containing one or more nitrogens;    -   or a pharmaceutically acceptable salt, hydrate or solvate        thereof.

In one embodiment, the present invention provides a compound of formulaI wherein A and D are a bond and B is C═O. In another embodiment, thepresent invention provides a compound of formula I wherein A is a bond,B is CH₂ are C is C═O. In another embodiment, the present inventionprovides a compound of formula I wherein Q is

In another embodiment, the present invention provides a compound offormula I wherein Z is

In another embodiment, the present invention provides a compound offormula I wherein one of R_(5a) and R_(5b) is hydrogen and the other isbenzyl. In another embodiment, the present invention provides a compoundof formula I wherein one of R_(5a) and R_(5b) is hydrogen and the otheris hydroxybenzyl. In another embodiment, the present invention providesa compound of formula I wherein one of R_(6a) and R_(6b) is hydrogen andthe other is benzyl. In another embodiment, the present inventionprovides a compound of formula I wherein one of R_(6a) and R_(6b) ishydrogen and the other is hydroxybenzyl. In another embodiment, thepresent invention provides a compound of formula I wherein one of R_(7a)and R_(7b) is hydrogen and the other is benzyl. In another embodiment,the present invention provides a compound of formula I wherein one ofR_(7a) and R_(7b) is hydrogen and the other is hydroxybenzyl. In anotherembodiment, the present invention provides a compound of formula Iwherein R₈ is benzyloxycarbonyl. In another embodiment, the presentinvention provides a compound of formula I wherein one of R_(9a) andR_(9b) is hydrogen and the other is benzyloxycarbonyl. In anotherembodiment, the present invention provides a compound of formula Iwherein L is NH₂.

In another embodiment, the present invention provides a heterocycliccompound represented by the structure of any of formulas II-X.

In one embodiment, the present invention provides a compound of formulaII.

In another embodiment, the present invention provides a compound offormula III.

In another embodiment, the present invention provides a compound offormula IV.

In another embodiment, the present invention provides a compound offormula V.

In another embodiment, the present invention provides a compound offormula VI.

In another embodiment, the present invention provides a compound offormula VII.

In another embodiment, the present invention provides a compound offormula VIII.

In another embodiment, the present invention provides a compound offormula IX.

In another embodiment, the present invention provides a compound offormula X.

As contemplated herein, the following definitions are used herein todescribe the compounds of the present invention:

The term “substituent” refers to a chemical radical or functional groupwhich is bonded to or incorporated onto the ring during the syntheticprocess used to generate the library. The different functional groupsare typically selected based on the knowledge concerning thepharmacophore that possesses a desired structure, function andbiological activity

The term “alkyl” refers to a straight or branched chain or cyclichydrocarbon having 1-12 carbon atoms. In one embodiment, the alkyl has1-10 carbons. In another embodiment, the alkyl has 1-8 carbons. Inanother embodiment, the alkyl has 1-6 carbons. In another embodiment,the alkyl has 1-4 carbons. The alkyl may be unsubstituted or by one ormore inert substituents, i.e. substituents which do not interfere withthe biological activity or conformational flexibility of the compounds.Non-limiting examples of suitable inert substituents are but are notlimited to halo, hydroxy, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,C₁-C₁₀ alkoxy, C₇-C₁₂ aralkyl, C₇-C₁₂ alkaryl, C₁-C₁₀ alkylthio,arylthio, aryloxy, arylamino, C₃-C₁₀ cycloalkyl, C₃-C₁₀ cycloalkenyl,di(C₁-C₁₀)-alkylamino, C₂-C₁₂ alkoxyalkyl, C₁-C₆ alkylsulfinyl, C₁-C₁₀alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy(C₁-C₁₀)alkyl,aryloxy(C₁-C₁₀)alkyl, C₁-C₁₀ alkoxycarbonyl, aryloxycarbonyl,aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano,cyano(C₁-C₁₀)alkyl, C₁-C₁₀ alkanamido, aryloylamido, arylaminosulfonyl,sulfonamido, amidino, amido, alkylamido, dialkylamido, amino,alkylamino, dialkylamino, carbonyl, carbamido, carboxy, heterocyclicradical, nitroalkyl, and —(CH₂)_(m)-Z-(C₁-C₁₀ alkyl), where m is 1 to 8and z is oxygen or sulfur.

The term “aryl” refers to an aromatic group having at least onecarbocyclic aromatic group, which may be unsubstituted or substituted byone or more inert substituents as defined hereinabove.

The term “heterocyclyl” or “heteroaryl” refers to a ring containing oneor more heteroatoms, for example oxygen, nitrogen, sulfur and the like,with or without unsaturation or aromatic character, optionallysubstituted with one or more inert substituents as defined hereinabove.Non-limiting examples of heterocyclic substituents are imidazole,pyrazole, pyrazine, thiazole, thiazine, oxazole, furan, dihydrofuran,tetrahydrofuran, pyridine, dihydropyridine, tetrahydropyridine,isoxazole and the like. Multiple rings may be fused, as in quinoline orbenzofuran, or unfused as in 4-phenylpyridine.

The heterocyclic moiety

is a heterocyclic moiety containing one or more nitrogens, which may beisolated or fused, for example and without being limited to—imidazole,pyrazole, pyrazine, pyridine, dihydropyridine, tetrahydropyridine,isoxazole, quinoline, isoquinoline and the like.

A “haloalkyl” group refers to an alkyl group as defined above, which issubstituted by one or more halogen atoms, e.g. by F, Cl, Br or I. A“hydroxyl” group refers to an OH group. An “alkenyl” group refers to agroup having at least one carbon to carbon double bond. A halo grouprefers to F, Cl, Br or I. An “arylalkyl” group refers to an alkyl boundto an aryl, wherein alkyl and aryl are as defined above. An example ofan arylalkyl group is a benzyl group.

The term “solid support” refers to a solvent insoluble material havingcleavable covalent bonds for use in preparing the library compounds ofthe invention.

As contemplated herein, the present invention further encompassesanalogs, derivatives, isomers, pharmaceutically acceptable salts andhydrates of the heterocyclic compounds defined by the present invention.

The term “isomer” includes, but is not limited to, optical isomers andanalogs, structural isomers and analogs, conformational isomers andanalogs, and the like. In one embodiment, this invention encompasses ofvarious optical isomers of the compounds of the present invention. Itwill be appreciated by those skilled in the art that the compounds ofthe present invention contain at least one chiral center. Accordingly,the these compounds exist in, and be isolated in, optically-active orracemic forms. Some compounds may also exhibit polymorphism. It is to beunderstood that the present invention encompasses any racemic,optically-active, polymorphic, or stereroisomeric form, or mixturesthereof. In one embodiment, the compounds the pure (R)-isomers. Inanother embodiment, the compounds are the pure (S)-isomers. In anotherembodiment, the compounds are a mixture of the (R) and the (S) isomers.In another embodiment, the compounds are a racemic mixture comprising anequal amount of the (R) and the (S) isomers. It is well known in the arthow to prepare optically-active forms (for example, by resolution of theracemic form by recrystallization techniques, by synthesis fromoptically-active starting materials, by chiral synthesis, or bychromatographic separation using a chiral stationary phase).

The invention includes pharmaceutically acceptable salts of theheterocyclic compounds of the present invention. Pharmaceuticallyacceptable salts can be prepared by treatment with inorganic bases, forexample, sodium hydroxide or inorganic/organic acids such ashydrochloric acid, citric acids and the like.

This invention further includes derivatives of the compounds. The term“derivatives” includes but is not limited to ether derivatives, acidderivatives, amide derivatives, ester derivatives and the like. Inaddition, this invention further includes hydrates of the compoundsdescribed herein. The term “hydrate” includes but is not limited tohemihydrate, monohydrate, dihydrate, trihydrate and the like.

Combinatorial Libraries

The invention also provides, according to another of its embodiments, acombinatorial library comprising a plurality of heterocyclic compoundshaving a ring size of between 8-20 atoms wherein the members of thelibrary differ from each other in the size of the ring, the chirality ofthe substituents on the ring or a combination of the two. In furtherpreferred embodiments the members of the library are distinct from oneanother in the nature, number, positioning, of substituents along thering, or in the nature and chirality of the linking moieties that jointhe subtituents to the ring.

Currently more preferred compounds according to the present inventionare represented by the structure of any of formulas I-X. A “library” isa collection of compounds which, while sharing some common structuralelements (which may be common scaffolds, common ring sizes, commonsubstituents and the like), are diverse from each other by at least oneof the following properties: i) the size of the ring; ii) the order inwhich the pharmacophores are arranged in the ring; iii) the chemicalnature of the ring; iv) the chemical nature of the pharmacophores; v)the chirality of the linker between the ring and the pharmacophore; andvi) the chirality of the pharmacophore. The library allows screeningfrom among a plurality of compounds for those that have a desiredproperty. The library may be designed by a combinatorial or classicalchemical process.

In one embodiment, the library comprises at least one compoundrepresented by the structure of formula I. In another embodiment, thelibrary comprises at least one compound represented by the structure offormula II. In another embodiment, the library comprises at least onecompound represented by the structure of formula III. In anotherembodiment, the library comprises at least one compound represented bythe structure of formula IV. In another embodiment, the librarycomprises at least one compound represented by the structure of formulaV. In another embodiment, the library comprises at least one compoundrepresented by the structure of formula VI. In another embodiment, thelibrary comprises at least one compound represented by the structure offormula VII. In another embodiment, the library comprises at least onecompound represented by the structure of formula VIII. In anotherembodiment, the library comprises at least one compound represented bythe structure of formula IX. In another embodiment, the librarycomprises at least one compound represented by the structure of formulaX.

In a preferred embodiment of the present invention, each member of thelibrary differs from the other by the size of the ring. In anotherpreferred embodiment, each member of the library differs from the otherby the chirality of the substituents on the ring. In another embodiment,each member of the library differs from the other by at least one of thesize of the ring or the chirality of the substitutents of the ring; andfurther differs by at least one of the chemical nature of the ring, thesubstituents on the ring, the linkers connecting the substituents andthe ring, the arrangement of the substituents on the ring, or anycombination thereof.

The combinatorial libraries of the invention serve as a readilyaccessible source of diverse macrocyclic compounds for use inidentifying new biologically active macrocyclic compounds throughpharmaceutical and veterinary candidate screening assays, for thedevelopment of highly effective and environmentally friendly insectcontrol and crop control agents, for use in studies definingstructure/activity relationships, and/or for use in clinicalinvestigation.

Design of Libraries and Identification of Biologically Active Compounds

Furthermore, in another embodiment, the present invention providesmethods for designing new compound libraries that have a novel type ofstructural complexity and diversity, and that can be screened toidentify potent compounds for pharmaceutical, veterinary or agriculturaluse. Molecules having a molecular weight of up to about 1000 daltons,i.e. small molecules, are preferable.

Thus, in one embodiment, the present invention provides a method ofidentifying a compound having a beneficial biological activity, by (a)designing a combinatorial library comprising a plurality of heterocycliccompounds having a ring size of between 8 to 20 atoms wherein somemembers of the library differ from others in at least one aspectselected from the ring size; and the chirality of the substituents onthe ring, wherein each member of the library comprises at least onepharmacophore potentially associated with the biological activity; (b)synthesizing a plurality of compounds from the combinatorial library;and (c) screening the synthesized compounds for candidates having thedesired biological activity.

In one embodiment, between step (a), i.e. planning the combinatoriallibrary, and step (b) i.e. synthesizing the compounds of the library, itis possible to add a step of virtually screening the library to identifythose compounds which most closely resemble or fit the pharmacophorethat interacts with a domain in the cellular component and is associatedwith the desired biological activity. Such virtual screening can helpand predict which compounds have a better chance of assuming thebioactive conformation and it is preferable to start the screening withthe compounds that are, according to 3D modeling the most likelymimics/docking compounds. Thus, in one embodiment, the method furthercomprises the following steps as part of the designing step (a): (a1)identifying a domain in a cellular component which is associated withthe biological activity; and (a2) virtually screening the combinatoriallibrary for lead compounds having a pharmacophore complementary to orcapable of mimicking the domain. In one embodiment, the virtualscreening step comprises virtual screening of the library with acomputer readable data storage material encoded with computer readabledata comprising three-dimensional structural determinants defining thedesired domain. In yet another aspect, the computer readable datastorage material is further encoded with a computer program logic forcontrolling a processor, wherein the computer program logic comprising aprocedure that enables the processor to identify a member of thecombinatorial library having a pharmacophore complementary to thedesired domain. domain. The above steps are showed schematically in thefollowing chart:

As the compounds of the invention are intended to mimic or to bind to adomain in a cellular component, for example a protein or a nucleic acid,so as to interrupt or to mimic its interaction with other cellularcomponents and thus modulate the biological activity (mediated by thecellular component ), it is desired that they resemble the desireddomain structurally and spatially as closely as possible. Therefore,when deciding at the library planning step how to produce the bestlibrary, and at the synthesizing step, which of the members of theplanned library should be synthesized, the following questions should beasked:

-   -   1. Are the relevant pharmacophores (or derivatives or mimics of        the pharmacophores) present in the planned library?    -   2. Is the order of the substituents on the ring and the distance        of the substituents from each other, suitable for achieving a        suitable bioactive conformation (correct positioning of the        pharmacophores)?    -   3. Is the compound capable in one of its conformations of        attaining the correct positioning of the pharmacophores?    -   4. Is the possible conformation energetically favorable?    -   5. Is there a certain degree of conformation flexibility to        allow conformational complementarity?

Most of the above questions can be answered during the planning stageand the synthesis decision stage on a computer using commerciallyavailable bioinformatic programs such as Tripose™.

The coordinates of amino acid side chains of a protein can be obtainedfrom the Protein Data Bank (PDB) files. This data is based on the 3Dstructure of the protein (preferably as a complex with the appropriateligand) either obtained by crystallography or homology modeling. The 3Dinformation allows to identify the exposed side chains and theseaccessible side chains are possible pharmacophores. In cases of proteinsfor which the 3D structure was not determined, the essential amino acidswithin a protein may be determined by the method of combinatorialalanine-scanning (Morrison and Weiss, (2001)), also known as Ala-Scan.Another method is known as omission libraries and is described inCampian et al. (1998). Yet other methods are site directed mutagenesisand protein engineering (Winter et al (1982)). The amino acids andbackbone elements essential for a certain function may be divided intotwo categories: those who interact with the receptive protein, nucleicacid, polysaccharide or cell membrane and those responsible for theconformation of the essential region. The side chains and backboneelements of the former are those that participate in the creation ofpharmacophores and the present invention relates to the creation of suchpharmacophores, or their mimics and their incorporation in the scaffoldsof the invention for the purpose of creating a mimic of a region of theprotein.

As mentioned above, the essential amino acids within a protein may bedetermined by the method of combinatorial alanine-scanning. Alaninescanning, a method of systematic and sequential alanine substitution,has been particularly useful for the identification of pharmacophores ina given peptide sequence. This method is based on the synthesis of alibrary in which each amino acid residue in a peptide chain issequentially replaced by alanine, and biological screening of thelibrary. Substitution of functional amino acid residues by the methylgroup of alanine leads to the removal of all the side chain atoms pastthe β-carbon. Thus, the role of side-chain functional groups at specificposition can be inferred. Alanine residue have the same backbonedihedral angles as other functional residues and thus the backboneconformation is not drastically perturbed by such substitution, as wouldbe the case in glycine scan libraries. In this case, the side chain isnullified, which leads to the introduction of flexibility into thepeptide backbone.

An additional method for the elucidation of pharmacophores is thesynthesis and biological screening of omission libraries. Omissionlibraries, based on a given peptide sequence is a library that containall the possible peptides that compose the parent peptide. Omissionlibrary is divided into sequential and non sequential. In sequentialomission library, amino acids are omitted from the carboxy- andamino-ends, whereas in non sequential library amino acids are omittedfrom the interior of the peptide sequence. Thus, sequential omissionlibrary based on a hexapeptide contains 2 pentapeptides, 3tetrapeptides, 4 tripeptides and 5 dipeptides (total of 14 peptides).Non-sequential omission library based on a hexapeptide contains 4pentapeptides, 18 tetrapeptides, 27 tripeptides and 9 dipeptides (totalof 58 peptides). Beside information on essential pharmacophores,omission libraries can furnish shorter active peptides that willfacilitate the design of libraries.

In conclusion, the method of the invention utilizes spatial librariesthat can generate novel leads for the disruption of protein:protein,protein:peptide, protein:cell membrane and protein:nucleic acidinteractions, in animals and plants.

Computer Automation

As contemplated herein, any of the methods provided by the presentinvention may be adapted for high-throughput by computer automation.Thus, the present invention provides computer program products virtualscreening of the combinatorial library of heterocyclic compoundsprovided herein for a compound having a beneficial biological activity.The computer program product according to one embodiment of the presentinvention comprises (a) a computer readable data storage materialencoded with computer readable data comprising three-dimensionalstructural determinants defining a domain in a cellular component whichis associated with the desired biological activity; and (b) a computerprogram logic for controlling a processor, comprising a procedure thatenables the processor to identify a compound having a pharmacophorecomplementary to the desired domain.

Furthermore, in yet another embodiment, the present invention provides asystem for identifying a compound having a beneficial biological,comprising: (a) an automated device for virtual screening of thecombinatorial library of heterocyclic compounds provided herein for acompound having said beneficial biological activity; and (b) anexperimental device for screening these compounds for candidates havingthe desired biological activity.

In one embodiment, the automated device comprises the computer programproduct described hereinabove. In another embodiment, the experimentaldevice comprises (a) an apparatus for synthesizing the compoundsidentified in step (a); and (b) an apparatus for experimentallyscreening the compounds for candidates having the desired biologicalactivity.

Pharmaceutical Compositions and Therapeutic Uses

In one embodiment, the compounds defined by the present invention areuseful in the treatment of a disease, disorder or condition. In anotherembodiment, therapy of the disease, disorder or condition is achieved bymodulation of a cellular component, for example a protein, a nucleicacid or a combination thereof. The compounds provided herein, eitheralone or in the pharmaceutical compositions are suitable for use in anysubject, for example a mammalian subject or a human subject. Thepharmaceutical compositions of the present invention are also suitablefor use in veterinary medicine and furthermore may be used inagriculture.

Furthermore, in another embodiment, the present invention provides apharmaceutical composition comprising at least one heterocyclic compoundas defined hereinabove, and a pharmaceutically acceptable carrier.Furthermore, in yet another embodiment, the present invention provides amethod for the treatment of a disease, condition or disorder in asubject in need thereof, comprising the step of administering to thesubject a therapeutically effective amount of a heterocyclic compoundprovided by the present invention.

The term “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen. For example this term may refer to an amountcapable of decreasing, to a measurable effect, at least one adversemanifestation of the disease and should be chosen in accordance with thedrug used, the mode of administration, the age and weight of thepatient, the severity of the disease, etc.

A “biological activity associated with a cellular component” refers to aphysiological property of a cell that is caused, directly or indirectly(the latter referring to an effect caused by an effector which is moredownstream in the pathway) by the interaction between one cellularcomponent to another (the term “cellular component” including: otherproteins or peptides of the same or different types, membranes, nucleicacids, lipoproteins, nucleotides, co-factors, hormones, ion effectorsand the like). The interaction between cellular components may be of thetype: receptor-ligand, enzyme-substrate, antigen-antibody, DNA-bindingproteins-DNA etc. Said interaction mediates (causes) directly orindirectly, a biological activity such as: expression of a protein,proliferation, differentiation, cell-elongation, cell-shape alteration,cellular metabolism, cellular uptake of external substances, secretionof substances from the cells and the like.

“Modulate/Modulator” refers to increase or decrease in at leastbiological activity associated with a cellular component, in thepresence of the compound of the invention, or to the change of theresponse of the cell to the presence of a physiological cue, as comparedto the activity or response, respectively, in the absence of thecompound. Examples of such physiological cues are presence of effectors,the modulation being a change in the cellular response to a ligand,hormone, response to toxic substances (pesticides), stress (heat shock,draught, lack of nutrients) aging and the like.

As contemplated herein, non-limiting examples of the desired biologicalactivity is proliferation, differentiation, phenotype alteration, uptakeof external substances into cells, secretion of substances from cells,metabolism, gene expression, protein expression, or any combinationthereof.

In accordance with another embodiment of the invention, the compounds ofthe invention may be bound to a detectable label such as afluorescence-emitting moiety, a radio-label, a label capable ofundergoing an enzymatic reaction producing a detectable color, a markerfor x-ray, MRI, radio-isotope imaging or PET scan, to produce a labeledadduct. Then, upon administration of such labeled adduct, it may bedetected at a desired location by any manner known in the art and inaccordance with the specific label used, for example, fluorescence,radioactive emission, or a color production, MRI, x-ray and the like.

The term “bound” refers to covalent or non-covalent (e.g.,electrostatic) binding, which connects the compound of the invention tothe detectable label. Alternatively, the compound of the invention mayhave inherent detectable properties of its own, that enable it to bedetected by any of the above mentioned techniques.

The pharmaceutical composition of the invention may be administered byany of the known administration routes, inter alia, oral, intravenous,intraperitoneal, intramuscular, subcutaneous, sublingual, intraocular,intranasal or topical administration routes. Appropriate unit dosageforms of administration include the forms for oral administration, suchas tablets, capsules, powders, granulates and oral solutions orsuspensions and the forms for sublingual and buccal administration, theforms for parenteral administration useful for a subcutaneous,intramuscular or intravenous injection, as well as the forms for rectaladministration.

The carrier should be selected in accordance with the desired mode ofadministration and include any known components, e.g. solvents;emulgators, excipients, talc; flavors; colors, etc. The pharmaceuticalcomposition may comprise, if desired, also other pharmaceutically-activecompounds which are used to treat the disease, eliminate side effects oraugment the activity of the active component.

In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to about 5 times per day or alternatively, asa continuous infusion. A typical preparation will contain from about 5%to about 95% active compound (w1w). Preferably, such preparationscontain from about 20% to about 80% active compound. As the skilledartisan will appreciate, lower or higher-doses than those recited abovemay be required. Specific dosage and treatment regimens for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration; rate ofexcretion, drug combination, the patient's disposition to the diseasestate and the judgment of the treating physician. In general, thecompound is most desirably administered at a concentration level thatwill generally afford effective results without causing any harmful ordeleterious side effects.

The pharmaceutical composition may comprise, if desired, also otherpharmaceutically-active compounds which are used to treat the disease,eliminate side effects or augment the activity of the active component.

Synthetic Approach

In general, the compounds of the invention are prepared according to theroutes showed in Schemes 1-4 below. The solid phase synthesis ofscaffolds I-IV comprise of a series of couplings of the appropriateprotected acids and reductive alkylations with ω-functionalizedprotected aldehydes. The assembly of the appropriate linear scaffold onthe solid support is followed by removal of the protecting groups P₁ andP₂ and cyclization. The appropriate scaffold is obtained afterdeprotection-removal from the solid support.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way,however, be construed as limiting the broad scope of the invention.

Experimental Details Section EXAMPLE 1

A library composed of 26 molecules that have the formula I wherein R¹and R² are either benzyl (side chain of phenylalanine) or hydroxybenzyl(side chain of tyrosine) and R³ is benzyloxycarbonyl (which is a mimicof the side chain of phenylalanine) was synthesized and characterized.The library was synthesized by the Simultaneous Multiple Solid Phasemethodology (Houghten (1985)) as showed in Scheme 5 below. The moleculeswere characterized by HPLC, MS and MS-MS spectrometry.

Synthetic Procedures According to Scheme 5 Above

Rink amide MBHA resin [Rink H. (1987)] (0.1 g in each bag, 0.6 mmol/g)was pre-swollen for 2 h in NMP while shaking in reaction vessel equippedwith sintered glass bottom. The Fmoc protecting group was removed fromthe resin by reaction with 20% piperidine in NMP (2×30 min). Fmocremoval was monitored by chloranil test. A coupling cycle was carriedout with Fmoc-AA (AA is abbreviation of amino acid) (5 eq), BTC (1.65eq), and 2,4,6 colidine (14 eq) in DCM for 2 h at room temperature.Reaction completion was monitored by qualitative chloranil test.Following coupling the peptidyl-resin was washed with DCM (×5) and for 2min. Fmoc removal and washing steps were carried out as described above.Fmoc removal was monitored by the chloranil test.

The peptidyl-resin was then washed by a mixture of NMP: MeOH 1:1/1% anda solution of the aldehyde (1 eq) in the mixture above was added (10 mlfor 12 bags). Then additional 40 ml of this mixture was added and themixture was shaken for 5 min. Then, 2 eq of NaBH₃CN were added and thereaction vessel was shaken for 2 h. The resin was washed as follows: DCM(2×2 min), EtOH (2×2 min), NMP (2×2 min), DCM (3×2 min). (Chloranil testgave blue color immediately). The following coupling was performed usingFmoc-AA (5 eq), BTC (1.65 eq) and 2,4,6 colidine (14 eq) indibromomethane at 50° C. for 2 h and was repeated when necessary. Fmocdeprotection and washing steps were carried out as described above.Reductive alkylation and washing steps were carried out as describedabove. A solution of benzylchloroformate (6 eq), and DIEA (12 eq) in DMFwas added to the resin and the mixture was shaken for 1 h. The reactionwas repeated and ten the resin was washed with NMP (5×2min) and DCM (2×2min). Reaction completion was monitored by chloranil test.

Disulfide Bridge Formation:

The disulfide bridge was oxidized using iodine (10 eq) in DCM andshaking for 3 h. The resin was washed as follows: DMF (2×2 min), 2%ascorbic acid in DMF (2×2 min), NMP (5×2 min), DCM (4×2 min).

Analytical Procedures:

All the crude compounds were analyzed by MS and analyticalreversed-phase HPLC (RP18 Vydak 4×250 mm; flow: 1 mL/min; T=30° C.;detection UV 214 nm; gradient: A=0.1% TFA in TDW, B=0.1% TFA in CH₃CN, 0min 95:5, 5 min 95:5, 33 min 5:95, 38 min 95:5, 42 min 95:5).

The molecules were purified by preparative reversed-phase HPLC (RP18Vydak 2.5×250 mm; flow: 9 mL/min; T=30° C.; detection UV 214 nm;gradient: A=0.1% TFA in TDW, B=0.1% TFA in CH₃CN, 0 min 95:5, 5 min95:5, 33 min 5:95, 38 min 95:5, 42 min 95:5).

Fractions were collected, lyophilized and characterized by analyticalHPLC and MS analysis. Results are shown in Table 1 below.

Synthesis of Aldehydes

Trityl thiopropanal:

a) Trityl-thiopropanoic acid

Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to a suspensionof NaH (11.5 g, 60% in mineral oil 0.288 mol) in 80 mL DMF under coolingand nitrogen atmosphere, the reaction mixture was stirred 30 minutesafter the addition was completed. Then, a solution of bromopropionicacid (20 g, 0.131 mol) dissolved in 50 mL DMF was added stepwise. Afterthe addition was completed the reaction mixture was stirred for 30minutes and then cooling and nitrogen atmosphere were stopped and thereaction mixture was sealed and left overnight. Then, 500 mL chloroformwere added and the mixture was washed with 4×200 mL of saturatedsolution of KHSO₄ and 4×200 mL TDW (the solid that precipitate duringthe washings should also be collected with the organic layer). Theorganic layer was evaporated and the product (that contained DMF traces)was precipitated by adding 300 mL TDW and stirring for few minutes. Theproduct was collected by filtration and dried by suction and then invacuo. The crude product was purified as follows: 150 mL of CHCl₃ wereadded to the white solid and the mixture was stirred for few minutes.Then 200 mL of PE 40-60 were added and the solid was collected byfiltration yielding 37.61 g (82% yield) of white powder, mp 177-183° C.,¹H NMR (CDCl₃, 300 MHz, 298K) δ 2.24 (t, 2H), 2.46 (t, 2H), 7.18-7.48(m, 15H). MS (ES) m/z 347.

b) Trityl-thiopropanoic acid hydroxamate

A solution of N,O dimethylhydroxylamine hydrochloride (2.188 g, 0.0225mol) in 40 mL DMF was added to a mixture of 6.96 g (0.02 mol) ofTrityl-thiopropanoic acid and PyBoP (11.45 g, 0.022 mol). DIEA (10.4 mL,0.06 mol) was added and the clear solution was stirred for 3 hours. EA(120 mL) was added to the stirred solution followed by 240 mL ofsaturated bicarbonate solution. The organic layer was collected andwashed with additional two portions of 100 mL of saturated bicarbonatesolution, 100 mL of TDW, 2×100 mL KHSO₄ 1M, and 100 mL TDW, dried overNa₂SO₄ and evaporated to dryness yielding 10.36 g (92% yield) of yellowoil. ¹H NMR (CDCl₃, 300 MHz) δ 2.38 (t, 2H), 2.51 (t, 2H), 3.10 (s, 3H),3.56 (s, 3H), 7.15-7.50 (m, 15H).

c) Trityl-thiopropanal

LiAlH₄ (2.014 g, 0.053 mol) was added in portions to a solution of 10.36g (0.0265 mol) of Trityl-thiopropanoic acid hydroxamate in 260 mL drydiethyl ether under cooling in ice bath and argon atmosphere. Thereaction mixture was stirred for 2 hours (monitored by TLC PE:EA=1:1).560 mL of EA were added followed by addition of 560 mL of KHSO₄ 1M. Themixture was stirred for additional 30 minutes. The organic layer wascollected and washed with 390 mL of KHSO₄ 1M and 390 mL of saturatedNaCl, dried over Na₂SO₄ and evaporated yielding 7.68 g (87% yield) ofwhite solid. ¹H NMR (CDCl₃, 300 MHz, 298K) δ 2.36 (t, 2H), 2.46 (t, 2H),7.15-7.50 (m, 15H), 9.55 (t, 1H).

Trityl thiobutyral

a) Trityl-thiobutyric acid:

Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to a suspensionof NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100 mL DMF undercooling and nitrogen atmosphere, the reaction mixture was stirred 30minutes after the addition was completed. Then, a solution ofbromobutyric acid (21.88 g, 0.131 mol) dissolved in 150 mL DMF was addedstepwise. After the addition was completed the reaction mixture wasstirred for 30 minutes and then cooling and nitrogen atmosphere werestopped and the reaction mixture was sealed and left overnight. Then,500 mL chloroform were added and the mixture was washed with 4×200 mL ofsaturated solution of KHSO₄ and 4×300 mL of water (the solid thatprecipitate during the washings should also be collected with theorganic layer). The organic layer was evaporated and the oily product(that contained DME traces) was triturated by adding 300 mL TDW andstirring vigorously for few minutes. The product was collected byfiltration, washed by TDW and dried by suction. The crude product waspurified as follows: 200 mL of PE was added to the white solid and themixture was stirred for 15 minutes. The solid was collected byfiltration and dried in vacuo yielding 36.82 g (77.6% yield) of whitepowder. ¹H NMR (CDCl₃, 300 MHz, 298K) δ 1.67 (m, 2H), 2.22 (t, 2H), 2.30(t, 2H), 7.10-7.50 (m, 15H).

b) Trityl-thiobutyric acid hydroxamate

A solution of N,O dimethylhydroxylamine hydrochloride (0.83 g, 0.0084mol) in 20 mL DMF was added to a mixture of 2.77 g (0.0076 mol) ofTrityl-thiobutyric acid and PyBoP (4.39 g, 0.0084 mol). DIEA (4 mL,0.023 mol) was added and the clear solution was stirred for 3 hours (pHshould be monitored and kept basic). EA (50 mL) was added to the stirredsolution followed by 90 mL of saturated bicarbonate solution. Theorganic layer was collected and washed with additional two portions of40 mL of saturated bicarbonate solution, 40 mL of water, 2×40 mL KHSO₄1M, and 40 mL water, dried over Na₂SO₄ and evaporated to drynessyielding 3.06 g (quantitative yield) of yellow oil. ¹H NMR (CDCl₃, 300MHz) δ 1.74 (m, 2H), 2.23 (t, 2H), 2.38 (t, 2H), 3.13 (s, 3H), 3.63 (s,3H), 7.10-7.50 (m, 15H).

c) Trityl-thiobutanal

LiAlH₄ (0.574 g, 0.0151 mol) was added in portions to a solution of 3.06g (0.0075 mol) of the hydroxamate in 100 mL dry diethyl ether undercooling in ice bath and argon atmosphere. The reaction mixture wasstirred for 1 hour (monitored by TLC PE:EA=1:1). 150 mL of EA were addedfollowed by addition of 150 mL of KHSO₄ 1M. The mixture was stirred foradditional 30 minutes. The organic layer was collected and washed with100 mL of KHSO₄ 1M and 100 mL of saturated NaCl, dried over Na₂SO₄ andevaporated yielding 1.90 g (73% yield) of white solid. ¹H NMR (CDCl₃,300 MHz, 298K) δ 1.66 (m, 2H), 2.22 (t, 2H), 2.38 (t, 2H) 7.15-7.50 (m,15H), 9.61 (t, 1H).

Trityl thiovaleric aldehyde

a) Trityl-thiovaleric acid:

Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to a suspensionof NaH (1.5 g, 60% in mineral oil 0.288 mol) in 100 mL DMF under coolingand nitrogen atmosphere, the reaction mixture was stirred 30 minutesafter the addition was completed. Then, a solution of bromovaleric acid(23.71 g, 0.131 mol) dissolved in 150 mL DMF was added stepwise. Afterthe addition was completed the reaction mixture was stirred for 30minutes and then cooling and nitrogen atmosphere were stopped and thereaction mixture was sealed and left overnight. Then, 500 mL chloroformwere added and the mixture was washed with 4×200 mL of saturatedsolution of KHSO₄ and 4×300 mL of water (the solid that precipitateduring the washings should also be collected with the organic layer).The organic layer was evaporated resulting in a solid product (thatcontained DMF traces). 300 mL TDW were added and the mixture was stirredvigorously for few minutes. The product was collected by filtration andpartially dried by suction. The crude product was purified as follows:200 mL of PE was added to the white solid and the mixture was stirredfor a few minutes. The solid was collected by filtration and dried invacuo yielding 45.06 g (91% yield) of white powder. ¹H NMR (CDCl₃, 300MHz, 298K) δ 1.41 (m, 2H), 1.58 (m, 2H), 2.18 (m, 4H), 7.15-7.45 (m,15H). MS (ES) m/z 376.

b) Trityl-thiovaleric acid hydroxamate

A solution of N,O dimethylhydroxylamine hydrochloride (0.7 g, 0.0071mol) in 16 mL DMF was added to a mixture of 2.45 g (0.0065 mol) ofTrityl-thiovaleric acid and PyBoP (3.73 g, 0.0071 mol). DIEA (3.4 mL,0.02 mol) was added and the clear solution was stirred for 3 hours (pHshould be monitored and kept basic). EA (50 mL) was added to the stirredsolution followed by 90 mL of saturated bicarbonate solution. Theorganic layer was collected and washed with additional two portions of40 mL of saturated bicarbonate solution, 40 mL of water, 2×40 mL KHSO₄1M, and 40 mL water, dried over Na₂SO₄ and evaporated to drynessyielding 3.06 g (quantitive yield) of yellow oil. ¹H NMR (CDCl₃, 300MHz) δ 1.74 (m, 2H), 2.23 (t, 2H), 2.38 (t, 2H), 3.13 (s, 3H), 3.63 (s,3H), 7.10-7.50 (m, 15H).

c) Trityl thiovaleric aldehyde

LiAlH₄ (0.574 g, 0.0151 mol) was added in portions to a solution of 3.06g (0.0075 mol) of the hydroxamate in 100 mL dry diethyl ether undercooling in ice bath and argon atmosphere. The reaction mixture wasstirred for 1 hour (monitored by TLC PE:EA=1:1). 150 mL of EA were addedfollowed by addition of 150 mL of KHSO₄ 1M. The mixture was stirred foradditional 30 minutes. The organic layer was collected and washed with100 mL of KHSO₄ 1M and 100 mL of saturated NaCl, dried over Na₂SO₄ andevaporated yielding 1.90 g (73% yield) of white solid. ¹H NMR (CDCl₃,300 MHz, 298K) δ 1.66 (m, 2H), 2.22 (t, 2H), 2.38 (t, 2H) 7.15-7.50 (m,15H), 9.61 (t, 1H).

Trityl thiohexanal:

a) Trityl-thiohexanoic acid:

Trityl mercaptan (36.13 g, 0.131 mol) was added stepwise to a suspensionof NaH (11.5 g, 60% in mineral oil 0.288 mol) in 100 mL DMF undercooling and nitrogen atmosphere, the reaction mixture was stirred 30minutes after the addition was completed. Then, a solution ofbromohexanoic acid (25 g, 0.128 mol) dissolved in 150 mL DMF was addedstepwise. After the addition was completed the reaction mixture wasstirred for 30 minutes and then cooling and nitrogen atmosphere werestopped and the reaction mixture was sealed and left overnight. Then,500 mL chloroform were added and the mixture was washed with 4×200 mL ofsaturated solution of KHSO₄ and 4×300 mL of water (the solid thatprecipitate during the washings should also be collected with theorganic layer). The organic layer was evaporated and the product (thatcontained DMF traces) was triturated by adding 300 mL TDW and stirringvigorously for few minutes. The product was collected by filtration,washed by TDW and dried by suction. The crude product was purified asfollows: The solid was dissolved in a mixture of 150 ml of CHCl₃ and 200ml of PE, and the solution was evaporated. The oil obtained wastriturated by addition of 100 ml PE and 50 ml of Et₂O then 50 ml Et₂Oand 50 ml PE. The solid was collected by filtration and dried in vacuoyielding 33.92 g (68% yield) of white powder. ¹H NMR (CDCl₃, 300 MHz,298K) δ 1.31 (m, 2H), 1.37 (m, 2H), 1.50 (m, 2H), 2.15 (t, 2H), 2.26 (t,2H), 7.15-7.50 (m, 15H).

b) Trityl-thiohexanoic acid hydroxamate

A solution of N,O dimethylhydroxylamine hydrochloride (1.23 g, 0.0126mol) in 25 mL DMF was added to a mixture of 4.47 g (0.0115 mol) ofTrityl-thiohexanoic acid and PyBoP (6.56 g, 0.0126 mol). DIEA (6 mL,0.0344 mol) was added and the clear solution was stirred for 3 hours (pHshould be monitored and kept basic). EA (70 mL) was added to the stirredsolution followed by 130 mL of saturated bicarbonate solution. Theorganic layer was collected and washed with additional two portions of60 mL of saturated bicarbonate solution, 60 mL of water, 2×60 mL KHSO₄1M, and 60 mL water, dried over Na₂SO₄ and evaporated to drynessyielding 4.29 g (86% yield) of yellow oil. ¹H NMR (CDCl₃, 300 MHz) δ1.29 (m, 2H), 1.44 (m, 2H), 1.51 (m, 2H), 2.16 (t, 2H), 2.33 (t, 2H),3.15 (s, 3H), 3.65 (s, 3H) 7.15-7.50 (m, 15H).

c) Trityl-thiohexanal

LiAlH₄ (0.75 g, 0.0198 mol) was added in portions to a solution of 4.29g (0.0099 mol) of the hydroxamate in 130 mL dry diethyl ether undercooling in ice bath and argon atmosphere. The reaction mixture wasstirred for 1 hour (monitored by TLC PE:EA=1:1). 200 mL of EA were addedfollowed by addition of 200 mL of KHSO₄ 1M. The mixture was stirred foradditional 30 minutes. The organic layer was collected and washed with140 mL of KHSO₄ 1M and 140 mL of saturated NaCl, dried over Na₂SO₄ andevaporated yielding 3.45 g (93% yield) of white solid. ¹H NMR (CDCl₃,300 MHz, 298K) δ 1.38 (m, 2H), 1.49 (m, 2H), 1.60 (m, 2H), 2.15 (t, 2H),2.34 (t, 2H), 7.10-7.55 (m, 15H), 9.71 (t, 1H).

Trityl thioacetaldehyde and trityl thiopentanal were prepared byprocedures similar to those described above. TABLE 1 structure and MScharacterization of a library according to the present invention, thepreparation of which is showed in Scheme 5 above. compound M.W number R⁶R⁷ R⁸ m n M.W calc. Obsvd. 1 L-hydroxy L-Benzyl Z# 4 5 649.87 653.3benzyl 2 L-hydroxy L-Benzyl Z 5 4 649.87 653.3 benzyl 3 L-BenzylD-hydroxy Z 4 5 649.87 653.2 benzyl 4 L-Benzyl D-hydroxy Z 5 4 649.87653.3 benzyl 5 D-hydroxy L-Benzyl Z 4 5 649.87 653.2 benzyl 6 D-hydroxyL-Benzyl Z 5 4 649.87 653.2 benzyl 7 D-Benzyl L-hydroxy Z 4 5 649.87653.3 benzyl 8 D-Benzyl L-hydroxy Z 5 4 649.87 653.2 benzyl 9 L-hydroxyD-Benzyl Z 4 5 649.87 653.2* benzyl 10 L-hydroxy D-Benzyl Z 5 4 649.87653.3 benzyl 11 L-hydroxy L-hydroxy Z 6 6 707.94 711.79 benzyl benzyl 12D-hydroxy D-hydroxy Z 6 6 707.94 711.85 benzyl benzyl 13 L-hydroxyD-hydroxy Z 6 6 707.94 711.91 benzyl benzyl 14 D-hydroxy L-hydroxy Z 6 6707.94 711.43 benzyl benzyl 15 L-hydroxy L-Benzyl Z 6 6 691.94 694.8benzyl 16 D-hydroxy D-Benzyl Z 6 6 691.94 693.41 benzyl 17 L-hydroxyD-Benzyl Z 6 6 691.94 693.79 benzyl 18 D-hydroxy L-Benzyl Z 6 6 691.94693.91 benzyl 19 L-Benzyl L-hydroxy Z 6 6 691.94 693.79 benzyl 20D-Benzyl D-hydroxy Z 6 6 691.94 693.6 benzyl 21 L-Benzyl D-hydroxy Z 6 6691.94 693.23** benzyl 22 D-Benzyl L-hydroxy Z 6 6 691.94 693.23**benzyl 23 L-Benzyl L-Benzyl Z 6 6 675.95 677.24** 24 D-Benzyl D-Benzyl Z6 6 675.95 677.23** 25 L-Benzyl D-Benzyl Z 6 6 675.95 N.D 26 D-BenzylL-Benzyl Z 6 6 675.95 677.30**#Z = benzyloxy carbonyl*The peak was obtained relatively with low intensity.**The peak was analyzed with HRMS.

Mass spectrometric analysis: The discrepancy between the calculated andthe observed mass as described in Table 1 ranges between 1.5 to 2.5 amu.These results may indicate the existence of reduced non cyclic moleculerather then the oxidized desired macrocycles. In order to negate thispossibility the peaks were analyzed by splitting (marked with ** inTable 1). This analysis yielded the expected MW values with adiscrepancy of only 0.3 amu. Furthermore, these molecules were alsoanalyzed by MS-MS and a fragment indicating a disulfide bridge wasfound:

EXAMPLE 2 Biological Activity of Heterocyclic Compounds Having IGF-IReceptor

Many cancers are known to be associated with abnormal (over) activity ofthe IGF-1R receptor. Therefore inhibiting the phosphorylation of thiskinase is a promising approach for selectively inhibiting cancer cellproliferation.

Three amino acids in the activation loop of IGF-1 receptor wereidentified, by structural analysis, to be important for substratebinding. These amino acids are Tyr in position 1135, Tyr 1163 and Tyr1131—corresponding to positions 1162, 1163 and 1158, respectively inIRK. It was hypothesized that mimicking these amino acids would resultin an interruption of the kinase-substrate interaction and thus inhibitIGF-1R-dependent phosphorylation. A small library of compounds based onthe compound of formula:

was synthesized with amino acid residue mimicking Tyr and Phe positionedon rings of varying sizes, in different orders and having differentchiralities (Tyr, D-Tyr Phe or D-Phe). The ring structures weresubstituted with carbobenzoxy at position R₃ (mimicking Tyr at position1135 in IGF1-R) and Tyrosine side chain at position R₁, mimicking Tyr atposition 1136. The library of compounds is depicted in Table 2: TABLE 2Inhibition of MCF7 Inhibition proliferation Compound of IGF-1R (% ofname R₂ R₁ n m phosphorylation control) SIB1 Phe Tyr 4 5 − 62 SIB2 PheTyr 5 4 − 100 SI3 DTyr Phe 4 5 SIB4 DTyr Phe 5 4 − 100 SIB5 Phe DTyr 45 + 50 SIB6 Phe DTyr 5 4 + 100 SIB7 Tyr DPhe 4 5 + 31 SIB8 Tyr DPhe 5 4−/+ 100 SIB9 DPhe Tyr 4 5 + 55 SIB10 DPhe Tyr 5 4 + 90

The compounds were screened for their activity in inhibition of theproliferation of breast cancer cell line MCF7. Crude mixtures comprisingcompounds SIB1, SIB7 and to SIB10 were found to be active in inhibitionof proliferation, with SIB7 having the formula given bellow being themost active.

A compound termed “SIB7” was found to be active in inhibiting theproliferation of several cancer cell lines and was found to inhibitIGF-1 dependent phosphorylation of both the IGF-1 receptor itself (whichserves as its own substrate in trans-phosphorylation) and the downstreamelement ERK.

A stick model of the active compound SIB7, superimposed on the aminoacids of the IGF-1 activation loop (FIG. 1) shows that the compound SIB7can faithfully mimic at least the two amino acids Tyr at position 1135and Tyr at position 1136.

A FlexX run on the TRIPOSE™ software (FIG. 2) indicated that compoundSIB7 can dock into the binding site of IGF-1R, thus showing that it iscapable of preventing the trans-phosphorylation of one IGF-1R moleculeby another.

EXAMPLE 3 Inhibition of IGF-1 Receptor and ERK Phosphorylation by theCompound of the Invention SIB7

Western Blot Analysis

MCF-7 cells were starved in a serum-free medium for 14 h and exposed tovarying concentrations of SIB7 (2.5-100 uM dissolved in DMSO) for thelast 5 h of starvation. Cells were stimulated with IGF-1 (50 ng/ml) for10 minutes and lysed with lysis buffer containing 20 mM Tris HCl (pH7.5), 10% Glycerol, 1mM EDTA, 1 mM EGTA, 1% TritonX100, 0.5 mM Na₃VO₄,10 mM β-glycerophosphate, 5 mM NaPPi, 50 mM NaF, 1 mM benzamidine andprotease inhibitor cocktail (Sigma). Equal amounts of protein (25 μg)were separated by 8% SDS-polyacrylamide gel electrophoresis, and theresolved proteins were electrotransferred to nitrocellulose membrane.Membranes were incubated with 3% BSA in TBST (25 mM Tris-HCl (pH 7.4),0.17M NaCl, and 0.2% Tween 20) for 1 h at room temperature followed byincubation with primary antibodies (see below) at 4° C. overnight.Membranes were then washed with TBST and probed with HRP-conjugatedsecondary antibodies at room temperature for 1 h. Membranes were washedseveral times with TBST and visualized using enhanced chemiluminescencekit (Supper signal, Pierce).

The respective phosphorylation of IGF-1R and ERK was determined byWestern blotting using anti-phosphoIGF-1R (Tyr 1131)/Insulinreceptor(Tyr 1146) antibody (Cell signaling) andanti-diphospho(Thr183/Tyr185)ERK1&2 (Sigma). Blots were stripped andreprobed with anti-IGF1R/IR and anti-ERK (Santa Cruz biotechnology),respectively. Quantification of the blots was preformed using the ImageJ analysis system. Histograms represent the ratio between the levels ofthe phosphorylated protein (pIGF-1R and pERK) and the levels of thetotal proteins (IGF-1R and ERK), respectively.

The results are shown in FIG. 3A and FIG. 3B. As can be seen the ratioof both the phosphorylated IGF-1 receptor to non-phosphorylated receptor(FIG. 3A) as well as the ratio of phosphorylated ERK tonon-phosphorylated ERK (FIG. 3B), decreased, in a dose-dependent manner,with increasing concentration of the compound SIB7. This resultindicates that SIB7 was capable of interfering with thetrans-phosphorylation of the IGF-1 receptor itself and of interferingwith the phosphorylation of its down-stream elements—ERK, probably dueto interruption of the kinase-substrate interaction, due to docking ofthe compound in lieu of the native substrate (which may be either theERK or another IGF-1R molecule) in the substrate binging site of IGF-1R,thus inhibiting the phosphorylation or trans-phosphorylation.

EXAMPLE 4 Inhibition of Proliferation of Tumor Cells by SIB7

The following Human solid tumors cell lines, which proliferation isknown to be dependent on the phosphorylation activity of IGF1R, wereused: MDA231 (estrogen receptor deficient human breast cancer), MCF-7(estrogen receptor positive human breast cancer), PC3 and DU145 (hormonerefractory prostate cancer cells) , colon cancer cell line HT29 andPANC1 (pancreas cancer cells) were obtained from the American TypeCulture Collection. These cell lines were grown in RPMI 1640 mediumsupplemented with penicillin (100 U/ml), streptomycin (100 μg/ml),glutamine (2 mM) and 10% endotoxin free fetal calf serum (Hyclone).

A suspension of the cells at 2×10⁴ cells/ml was prepared in theabove-described culture mediums and distributed 0.180 ml per well (about4000 cells/well) in the wells of 96 well, flat bottom, tissue culturemicrotiter plates.

A series of compound stock solutions were prepared by diluting a 10 mMsolution of the compound in 100% DMSO with phosphate buffered saline(PBS) containing 0.1% bovine serum albumin (BSA) to a concentration of400 μM. The concentration of compound in each stock solution wasadjusted to ten times the desired concentration of the compound in theassay mixture. 0.020 ml of each compound stock solution was added to thecorresponding wells about 3 hours after cell addition, with threereplicates for each concentration. In addition, PBS containing 0.1% BSAsolution with either no added compound (NT) or final 0.1% DMSO was usedas a control. The plates were labeled and incubated for 72-80 hours at37° C. in a 5% CO₂ humidified incubator.

The medium discarded and the wells were fixed with 4% formaldehyde inPBS (formalin was obtained from Fisher Scientific; Catalog No. HC200-1)(0.2 ml/well) for at least 30 minutes. The wells were washed one timewith borate buffer (0.2 ml/well) (0.1M, pH 8.5). Freshly filtered 1%methylene blue solution (0.06 ml/well) was then added to the wells andincubated for 10 minutes at room temperature. The wells were then washedfive times with tap water, after which the wells were dried completely.0.20 ml/well of 0.1 N HCl was added to extract the color. Afterovernight extraction, the O.D. was read at 595 nm to determine thenumber of cells per well. The procedure for counting cells is describedin greater detail in Oliver et al, (1989), the teachings of which areincorporated herein by reference.

The proliferation results are shown in the attached Table 3 and in FIG.4 for proliferation of MCF-7 cell line.

As can be seen, SIB7, which was shown to inhibit IGF-1R-associatedphosphorylation (see above example) was capable of inhibiting theproliferation of a number of cancer cell lines, which proliferation isknown to be dependent on IGF-1R activity. TABLE 3 Inhibition of cancercell line proliferation by compound SIB7: Cell line IC₅₀ (μm) DU145 85PC3 48 HT29 — MCF7 16 MDA231 37 PANC1 42

It will be appreciated by a person skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed hereinabove. Rather, the scope of the invention is defined bythe claims which follow:

REFERENCES

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1. A heterocyclic compound represented by the structure of formula I:

wherein A, B and D are independently of each other CH₂, C═O or a bond; Xand Y are independently of each other O, S, C═O, S═O, SO₂,CR_(3a)R_(3b), NR₄ or C═S, or X and Y together form a group representedby the formula:

R_(1a), R_(1b), R_(2a), R_(2b), R_(3a), R_(3b), R_(4a) and R_(4b) areindependently of each other hydrogen or a linear or branched chainalkyl; m and n are independently of each other an integer of 1-6; Z andQ are independently of each other

R_(5a), R_(5b), R_(6a), R_(6b), R_(7a), R_(7b), R₈, R_(9a), and R_(9b)are independently of each other hydrogen, a linear or branched chainalkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, acyl,carboxyalkyl, carboxyaryl, benzyl, hydroxybenzyl, benzyloxycarbonyl, aside chain of a natural or unnatural amino acid or a peptide; L ishydrogen, OR₁₀, or NHR₁₁ wherein R₁₀ and R₁₁ are independently of eachother hydrogen, a linear or branched chain alkyl, a side chain of anatural or unnatural amino acid, a peptide or a solid support; and

is a heterocyclic moiety containing one or more nitrogens; or apharmaceutically acceptable salt, hydrate or solvate thereof.
 2. Thecompound according to claim 1, wherein A and D are a bond and B is C═O.3. The compound according to claim 1, wherein A is a bond, B is CH₂ areC is C═O.
 4. The compound according to claim 1, wherein Q is


5. The compound according to claim 1, wherein Z is


6. The compound according to claim 1, wherein one of R_(5a) and R_(5b)is hydrogen and the other is benzyl or hydroxybenzyl.
 7. The compoundaccording to claim 1, wherein one of R_(6a) and R_(6b) is hydrogen andthe other is benzyl or hydroxybenzyl.
 8. The compound according to claim1, wherein one of R_(7a) and R_(7b) is hydrogen and the other is benzylor hydroxybenzyl.
 9. The compound according to claim 1, wherein R₈ isbenzyloxycarbonyl.
 10. The compound according to claim 1, wherein one ofR_(9a) and R_(9b) is hydrogen and the other is benzyloxycarbonyl. 11.The compound according to claim 1, wherein L is NH₂.
 12. The compoundaccording to claim 1, represented by the structure of any of formulasII-X.


13. The compound according to claim 12, represented by the structure offormula II.
 14. The compound according to claim 12, represented by thestructure of formula III.
 15. The compound according to claim 12,represented by the structure of formula IV.
 16. The compound accordingto claim 12, represented by the structure of formula V.
 17. The compoundaccording to claim 12, represented by the structure of formula VI. 18.The compound according to claim 12, represented by the structure offormula VII.
 19. The compound according to claim 12, represented by thestructure of formula VIII.
 20. The compound according to claim 12,represented by the structure of formula IX.
 21. The compound accordingto claim 12, represented by the structure of formula X.
 22. The compoundaccording to claim 1, wherein said compound comprises at least onepharmacophore potentially associated with a biological activity.
 23. Thecompound according to claim 22, wherein said biological activity ismediated by a cellular component, wherein said cellular component is aprotein, a nucleic acid, or a combination thereof.
 24. The compoundaccording to claim 23, wherein said biological activity isproliferation, differentiation, phenotype alteration, uptake, secretion,metabolism, gene expression, protein expression, or any combinationthereof.
 25. A combinatorial library comprising a plurality of compoundsrepresented by the structure of formula I:

wherein A, B and D are independently of each other CH₂, C═O or a bond; Xand Y are independently of each other O, S, C═O, S═O, SO₂,CR_(3a)R_(3b), NR₄ or C═S, or X and Y together form a group representedby the formula:

R_(1a), R_(1b), R_(2a), R_(2b), R_(3a), R_(3b), R_(4a) and R_(4b) areindependently of each other hydrogen or a linear or branched chainalkyl; m and n are independently of each other an integer of 1-6; Z andQ are independently of each other

R_(5a), R_(5b), R_(6a), R_(6b), R_(7a), R_(7b), R₈, R_(9a), and R_(9b)are independently of each other hydrogen, a linear or branched chainalkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, acyl,carboxyalkyl, carboxyaryl, benzyl, hydroxybenzyl, benzyloxycarbonyl, aside chain of a natural or unnatural amino acid or a peptide; L ishydrogen, OR₁₀, or NHR₁₁ wherein R₁₀ and R₁₁ are independently of eachother hydrogen, a linear or branched chain alkyl, a side chain of anatural or unnatural amino acid, a peptide or a solid support; and

is a heterocyclic moiety containing one or more nitrogens; or apharmaceutically acceptable salt, hydrate or solvate thereof.
 26. Thelibrary according to claim 25, comprising at least one compound offormula I wherein A and D are a bond and B is C═O.
 27. The libraryaccording to claim 25, comprising at least one compound of formula Iwherein A is a bond, B is CH₂ are C is C═O.
 28. The library according toclaim 25, comprising a plurality of compounds represented by thestructure of any of formulas II-X.


29. The library according to claim 25, comprising a plurality ofcompounds represented by the structure of formula II.
 30. The libraryaccording to claim 25, comprising a plurality of compounds representedby the structure of formula III.
 31. The library according to claim 25,comprising a plurality of compounds represented by the structure offormula IV.
 32. The library according to claim 25, comprising aplurality of compounds represented by the structure of formula V. 33.The library according to claim 25, comprising a plurality of compoundsrepresented by the structure of formula VI.
 34. The library according toclaim 25, comprising a plurality of compounds represented by thestructure of formula VII.
 35. The library according to claim 25,comprising a plurality of compounds represented by the structure offormula VIII.
 36. The library according to claim 25, comprising aplurality of compounds represented by the structure of formula IX. 37.The library according to claim 25, comprising a plurality of compoundsrepresented by the structure of formula X.
 38. The library according toclaim 25, wherein each member of the library differs from the other bythe size of the ring, the chirality of the substituents on the ring, orby a combination thereof.
 39. The library according to claim 38, whereineach member of the library further differs from the other by thechemical nature of the ring, the substituents on the ring, the linkersconnecting the substituents and the ring, the arrangement of thesubstituents on the ring, or any combination thereof.
 40. The libraryaccording to claim 25, wherein each member of the library comprises atleast one pharmacophore potentially associated with a biologicalactivity.
 41. The library according to claim 40, wherein said biologicalactivity is mediated by a cellular component, wherein said cellularcomponent is a protein, a nucleic acid, or a combination thereof. 42.The library according to claim 41, wherein said biological activity isproliferation, differentiation, phenotype alteration, uptake, secretion,metabolism, gene expression, protein expression, or any combinationthereof.
 43. A method of identifying a compound having a beneficialbiological activity, said method comprising the steps of: designing acombinatorial library comprising a plurality of heterocyclic compoundsaccording to claim 1, wherein each member of the library comprises atleast one pharmacophore potentially associated with said biologicalactivity; synthesizing a plurality of compounds from said combinatoriallibrary; and screening said compounds for candidates having saidbiological activity.
 44. The method according to claim 43, wherein eachmember of the library differs from the other by the size of the ring,the chirality of the substituents on the ring, or by a combinationthereof.
 45. The method according to claim 43, wherein some members ofthe library differs from the others by the chemical nature of the ring,the substituents on the ring, the linkers connecting the substituentsand the ring, the arrangement of the substituents on the ring, or anycombination thereof
 46. The method according to claim 43, wherein saidbiological activity is achieved by modulation of a cellular component.47. The method of claim 46, wherein said pharmacophore is complementaryto a domain in said cellular component which is associated with saidbiological activity.
 48. The method according to claim 43, wherein saiddesigning step further comprises: identifying a domain in a cellularcomponent which is associated with said biological activity; andvirtually screening said combinatorial library for lead compounds havinga pharmacophore complementary to said domain.
 49. The method accordingto claim 48, wherein said virtual screening step comprises virtualscreening with a computer readable data storage material encoded withcomputer readable data comprising three-dimensional structuraldeterminants defining said domain.
 50. The method according to claim 49,wherein said computer readable data storage material is further encodedwith a computer program logic for controlling a processor, said computerprogram logic comprising a procedure that enables said processor toidentify a member of said combinatorial library having a pharmacophorecomplementary to said domain.
 51. The method according to claim 48,wherein said cellular component is a protein, a nucleic acid or acombination thereof.
 52. The method according to claim 43, wherein saidbiological activity is proliferation, differentiation, phenotypealteration, uptake, secretion, metabolism, gene expression, proteinexpression, or any combination thereof.
 53. A computer program productfor virtual screening of a combinatorial library of heterocycliccompounds according to claim 1 for a compound having a beneficialbiological activity, said computer program product comprising: acomputer readable data storage material encoded with computer readabledata comprising three-dimensional structural determinants defining adomain in a cellular component which is associated with said biologicalactivity; and p1 a computer program logic for controlling a processor,said computer program logic comprising a procedure that enables saidprocessor to identify a compound having a pharmacophore complementary tosaid domain.
 54. The computer program product according to claim 53,wherein said cellular component is a protein, a nucleic acid or acombination thereof.
 55. The computer program product according to claim53, wherein said biological activity is proliferation, differentiation,phenotype alteration, uptake, secretion, metabolism, gene expression,protein expression, or any combination thereof.
 56. The computer programproduct according to claim 53, wherein each member of the librarydiffers from the other by the size of the ring, the chirality of thesubstituents on the ring, or by a combination thereof.
 57. The computerprogram product according to claim 53, wherein each member of thelibrary differs from the other by the chemical nature of the ring, thesubstituents on the ring, the linkers connecting the substituents andthe ring, the arrangement of the substituents on the ring, or anycombination thereof
 58. A system for identifying a compound having abeneficial biological, comprising: an automated device for virtualscreening of a combinatorial library of heterocyclic compounds accordingto claim 1 for a compound having said beneficial biological activity;and an experimental device for screening said compounds for candidateshaving said biological activity.
 59. The system according to claim 58,wherein said automated device comprises a computer program productcomprising: a computer readable data storage material encoded withcomputer readable data comprising three-dimensional structuraldeterminants defining a domain in a cellular component which isassociated with said biological activity; and a computer program logicfor controlling a processor, said computer program logic comprising aprocedure that enables said processor to identify a compound having apharmacophore complementary to said domain.
 60. The system according toclaim 59, wherein said cellular component is a protein, a nucleic acidor a combination thereof. complementary to said domain.
 61. The systemaccording to claim 58, wherein said biological activity isproliferation, differentiation, phenotype alteration, uptake, secretion,metabolism, gene expression, protein expression, or any combinationthereof.
 62. The system according to claim 58, wherein some members ofthe library differ from the others by the size of the ring, thechirality of the substituents on the ring, or by a combination thereof.63. The system according to claim 58, wherein each member of the librarydiffers from the other by at least one additional feature selected from:the chemical nature of the ring, the substituents on the ring, thelinkers connecting the substituents and the ring, the arrangement of thesubstituents on the ring, or any combination thereof.
 64. The systemaccording to claim 58, wherein said experimental device comprises anapparatus for synthesizing said compounds; and an apparatus forscreening said compounds for candidates having said biological activity.65. A pharmaceutical composition comprising at least one compoundaccording to claim 1, and a pharmaceutically acceptable carrier.
 66. Thecomposition according to claim 65, for therapeutic use in the treatmentof a disease, disorder or condition.
 67. The composition according toclaim 66, wherein a therapeutic effect is achieved by modulation of acellular component, wherein said cellular component is a protein, anucleic acid or a combination thereof.
 68. The composition according toclaim 67, wherein the modulation of said cellular component affects abiological activity selected from proliferation, differentiation,phenotype alteration, uptake, secretion, metabolism, gene expression,protein expression, or any combination thereof.
 69. A method for thetreatment of a disease, condition or disorder in a subject in needthereof, comprising the step of administering to said subject atherapeutically effective amount of a compound according to claim
 1. 70.The method according to claim 69, wherein a therapeutic effect isachieved by modulation of a cellular component in said subject, whereinsaid cellular component, is a protein, a nucleic acid, or a combinationthereof.
 71. The method according to claim 70, wherein the modulation ofcellular component affects a biological activity selected fromproliferation, differentiation, phenotype alteration, uptake, secretion,metabolism, gene expression, protein expression, or any combinationthereof.
 72. A method of modulating a cellular component in a cell,comprising the step of contacting said cell with a compound according toclaim 1, in an amount effective to modulate said cellular component,wherein said cellular component is a protein, a nucleic acid, or acombination thereof.
 73. The method according to claim 72, wherein themodulation of said cellular component affects a biological activityselected from proliferation, differentiation, phenotype alteration,uptake, secretion, metabolism, gene expression, protein expression, orany combination thereof.
 74. A combinatorial library comprising aplurality of heterocyclic compounds, wherein the ring size of theheterocyclic compounds is between 8 and 20 atoms, and members of thelibrary differ from each other by at least one of: the size of the ring;and the chirality of the substituents on the ring; wherein the membersof the libraries comprise at least one common pharmacophore.
 75. Thelibrary according to claim 74 wherein the members of the library maydiffer from each other by at least one additional parameter selectedfrom the chemical nature of the ring; the order of the substituents onthe ring; the type of linker connecting the substituent to the ring.